The clinical success of macrolide antibiotics has been curbed due to spread of resistance. One of the major resistance mechanism found in pathogenic strains is modification of the drug-target, the ribosome, by Erm methyltransferase enzymes. The expression of many of the erm genes, including ermC, is inducible by macrolides. The proposed mechanism of induction is based on the programmed ribosome stalling at the regulatory cistron, ermCL. The newer generation of macrolides, ketolide antibiotics, were considered to be non-inducers of inducible erm genes. However, recent data have shown that ketolides do induce expression of ermC. Our preliminary data show that in contrast to erythromycin and macrolides of the second generation, ketolides do not induce ribosome stalling at ermCL. This and several other observations strongly suggest that the mechanism of erm induction by ketolides is principally different from that of macrolides. Unraveling this mechanism is the primary goal of this proposal. We propose three main possible models that potentially explain how ketolides induce erm expression. ln the 'slide-through'model, ketolides allow the ribosome translating ermCL to reach the initiation codon of ermC via frameshifting or stop codon read-through. In the 'hopping'model, ketolides promote the bypass the ermCL-ermC intergenic region and allow the ribosome resume translation at the erm cistron. The third, 'riboswitch', model suggests that the direct interaction of ketolide antibiotics with mRNA lead to a conformational switch that facilitates initiation of translation of the erm gene. We propose a series of genetic and biochemical experiments to test and discriminate between these models. Finally, we will analyze the possible induction of other clinically relevant erm genes by ketolide antibiotics. Understanding the mechanism of ketolide-dependent induction of the erm genes offers the opportunity to decipher a new molecular mechanism of regulation of expression of antibiotic resistance genes and will contribute to design new non- inducing antibiotics. PUBLIC HEALTH RELEVANCE: Ketolide antibiotics induce expression of resistance gene of clinically important bacterial pathogens. The goal of this project is to unravel this mechanism. Deciphering the way antibiotics turn on the expression of resistance genes at the molecular level will eventually lead to the design of new non-inducer antibiotics.